Systematic evaluation of phenyl stationary phases for oligonucleotide analysis without ion-pairing reagents

Oligonucleotides (ONs) represent a rapidly expanding class of therapeutics. Yet their analysis remains a challenge due to their highly charged backbone, extensive structural modifications, and complex impurity profiles. Ion-pair reversed-phase liquid chromatography (IP-RPLC) is the current gold standard for ON separations; however, the development of chromatographic methods that avoid ion-pairing reagents is highly desirable. In this study, we systematically evaluated a panel of phenyl-based stationary phases as an alternative approach for performing ion-pair free RPLC of ONs. Nine phenyl-modified columns, including phenyl-hexyl, diphenyl, biphenyl, pentafluorophenyl (PFP), and pentabromophenyl phases, were screened. Among them, the biphenyl and PFP columns demonstrated superior performance in terms of peak capacity and selectivity. Optimal separations were achieved using 50 mM ammonium acetate at pH 8, methanol as the organic modifier, a column temperature of 50 °C, a 20 min gradient, and a flow rate of 0.3 mL/min for a 2.1 mm column diameter. To better understand the retention mechanisms, chromatographic experiments were complemented with quantum mechanical modelling and circular dichroism measurements, confirming that retention and selectivity are primarily governed by π-π stacking in the presence of methanol, with interaction energies comparable to those of DNA base stacking. Notably, retention decreases markedly for ONs forming stable secondary structures (poly dC and poly dG) that hinder intermolecular stacking. Two applications illustrated the relevance of phenyl-based RPLC in the absence of ion pairing: analysis of linear and structured shortmers of a 20-mer antisense oligonucleotide (ASO), and characterization of a chemically modified ASO. These case studies highlighted the complementarity of biphenyl and PFP chemistries in terms of selectivity. Peak capacities were found to be comparable to those obtained with IP-RPLC using triethylamine, and retention increases as follows: IP-RPLC (TEAA) < biphenyl-RPLC < PFP-RPLC. Overall, phenyl-based stationary phases provide a robust, MS-compatible alternative to conventional ion-pairing methods, expanding analytical approaches for ON characterization.